Method for determining bacterial populations

ABSTRACT

A method for determining the population density of bacterial cells in an environment comprising, 1. separating substantially all nonbacterial cells from an aqueous sample of the environment, for example, by low force centrifugation, 2. separating substantially all bacterial cells from the sample, e.g., by filtration, 3. extracting adenosine triphosphate (hereinafter referred to as ATP) from the bacterial cells, 4. measuring the quantity of ATP present, such as, by reacting the ATP with luciferin and luciferase and measuring the light emitted, and 5. determining the number of bacterial cells in the sample, preferably, by dividing the quantity of ATP, in micrograms, by the average ATP/per cell which was found to be about 5 X 10 10 micrograms.

United States Patent [72] Inventor Anthony J. DEustachio Wilmington,Del.

[21] Appl. No. 635,109

[22] Filed May 1, 1967 [45] Patented Oct. 26, 1971 [73] Assignee E. I.du Pont de Nemonrs and Company Wilmington, Del.

[54] METHOD FOR DETERMINING BACTERIAL Rose, A. H. Chemical Microbiology"1965 pp. 194- 5 Schaechter et a1. J. Gen. Micro." 19: 592- 606 1958.

Framsen, .l. S. & Binkley, S. B. Comparison of the Acid- SolubleNucleotides in Escherichia Coli at different Growth Rates." JournalBiologicalChem. Feb. 1961 236: 515-518 Colowick et al., Methods InEnzymology Vol. III pp. 871- 873 (1957) Primary Examiner-A. LouisMonacell Assistant Examiner-Max D. Hensley Artomey-Herbert M. WolfsonABSTRACT: A method for determining the population density of bacterialcells in an environment comprising, 1. separating substantially allnonbacterial cells from an aqueous sample of the environment, forexample, by low force centrifugation, 2. separating substantially allbacterial cells from the sample, e.g., by filtration, 3. extractingadenosine triphosphate (hereinafter referred to as ATP) from thebacterial cells, 4. measuring the quantity of ATP present, such as, byreacting the ATP with luciferin and luciferase and measuring the lightemitted, and 5. determining the number of bacterial cells in the sample,preferably, by dividing the quantity of ATP, in micrograms, by theaverage ATP/per cell which was found to be about 5X10 micrograms.

METHOD FOR DETERMINING BACTERIAL POPULATIONS CROSS REFERENCES TO RELATEDAPPLICATIONS A detailed description of methods and apparatus fordetecting the presence of life based on the ubiquity of ATP in livingorganisms may be found in U.S. application Ser. No. 433,488, nowabandoned, filed Feb. 19, 1965, entitled Method of Detecting LivingOrganisms, and U.S. application Ser. No. 434,240, now U.S. Pat. No.3,359,973, filed Feb. 19, 1965, entitled Bioluminescence ReactionChamber. Additionally, U.S. application Ser. No. 550,103, now U.S. Pat.No. 3,432,487, filed May 16,1966, entitled Process for Extraction andConcentration of Hydrophilic Substances, discloses a method forextracting ATP from cellular organisms. These cross-references aremerely exemplary and are not intended to restrict the scope of thepresent invention as set forth in the appended claims.

BACKGROUND OF THE INVENTION ATP is present in all living organisms.Therefore, by analyzing a sample of an environment for ATP, it ispossible to detect the presumptive presence of living organisms in thatenvironment. This analysis is particularly useful for determining thepresence of bacteria in various environments. Often, how ever, aquantitative, rather than a qualitative, determination is required.Quantitative analysis for bacteria is desirable, for example, in thedetermination of background levels of bacteria in environments, such as,air, fuels, water, milk, food, medical supplies, pharmaceuticals, cleanassembly areas, hospital rooms and germJree areas, and the detection ofincreased contamination in these environments; in the detection ofinfections, e.g., kidney infections by urine analysis; in biomedicalstudies of bacteria cultures; and in monitoring the effectiveness ofpollution controls and sterilization procedures and the sterilization ofcompounds and apparatus. One way to obtain a quantitative determinationis by counting colonies of bacterial cells an agar plate cultures.However, this method is tedious, requiring considerable time and effortFurthermore, accuracy is limited by cell clumping.

SUMMARY OF THE INVENTION The instant invention provides a substantiallyrapid and accurate method for determining the quantitative existence ofbacteria in a given environment. This method comprises the followingsteps:

1. separating substantially all nonbacterial cells from an aqueoussuspension of a sample of an environment,

2. separating substantially all bacterial cells from said suspension,

3. extracting ATP from said bacterial cells, and

4. measuring the quantity of adenosine triphosphate extracted wherebythe population density of bacterial cells in said environment isdetermined.

For a detailed understanding of the invention, reference is made to thefollowing description of various embodiments thereof and to theattendant drawing which is a computer printout of data obtained inaccordance with one embodiment of the invention.

Biological material containing bacterial cells may be obtained, interalia, from animal tissues, suspensions of micro-organisms, blood, urine,water or beverages suspected of contamination, patient exudates, air orfuel suspected of containing micro-organisms, and food or othercontaminated environments. The procuring of a sample of an environmentand its subsequent storage prior to assay for ATP may be carried out byany appropriate method known in the art. The size of the sam plc iscontrolled by the sensitivity and range of the means used to measure thequantity of ATP present in the sample. The sample, it not already inaqueous form, may be suspended in sterile water, buffer or salinesolution. It is then treated to remove substantially all nonbacterialcells, e.g., living organisms, leucocytes, etc. This may be performed bycentrifugation at a force sufficient to result in a centrifugate havinga lower layer substantially free from bacterial cells and an upper layer(supernatant) substantially free from nonbacterial cells. This ispreferably carried out at a force from about x 3 to about 250x g.

After this separation, the supernatant (if the prior step was performedby centrifugation) containing bacterial cells is subjected to a furtherseparation step wherein the bacterial cells are isolated fromsubstantially all extracellular water-soluble material. The cells arepreferably isolated by passing the supernatant through a microporousmembrane and subsequently washing, e.g., with sterile cold salinesolution.

Applicant has found that the aforementioned extracellular water-solublematerial, including, for example, contaminating salts, acids andnonbacterial ATP, interferes with the accuracy of the subsequent ATPassay. In particular, the presence of soluble nonbacterial ATP leads tohigh, irregular values for intracellular ATP.

The indicated order of the previous two steps is desirable. Applicanthas found that effecting separation of water-soluble material prior tothe removal of nonbacterial cells may result in high values forintercellular ATP. Apparently this is a result of the presence ofresidual nonbacterial ATP in the assayed sample.

Qualitative results may be obtained by assaying directly the intactbacterial cells. However, for accurate quantitative assays, it isdesirable to extract ATP from the cells. Typical methods for carryingout this extraction include the use of hot water, acetone,dimethylsulfoxide, perchloric acid, butanol, ethyl alcohol, etc., andultrasonic disruption of the cells. This extraction is preferablyaccomplished by immersing the cells in a buffered aqueous solution ofn-butanol as described in application Ser. No. 550,103. The relativeamounts of nbutanol, and buffer solution are important. Sufficientn-butanol is used to bring about the release of ATP and sufficientbuffer solution is used to provide the desired volume of aqueous phasewhich carries the ATP released from the cells.

An aqueous extract of ATP may then be assayed by means of the fireflybioluminescent technique disclosed in application Ser. No. 433,488, bycontacting an aliquot of the extract in the presence of oxygen with areaction mixture which contains luciferin, luciferase and a magnesiumsalt, and monitoring for the emission of light. The aqueous reactionmedium (reaction mixture-l-extract) will generally contain enough oxygento allow the reaction to take place. The quantity or maximum intensityof light emitted is a measure of the intracellular ATP present. Byapplication of the single correlation shown in the accompanying drawing(later discussed), the population density of the bacterial cells may bedetermined.

The reaction mixture may be prepared using firefly lantern extract orthe individual constituents which participate in the bioluminescentreaction. Commercial lyophilized firefly lantern extract is preferablydissolved in a sterile aqueous solution (pH 7.4) having MgSO, andpotassium arsenate in concentrations of 0.01 M and 0.05 M, respectively.Alternatively, various other buffers, such as,tris(hydroxymethyl)aminomethane, may be used.

Firefly lantern extract may also be obtained in the laboratory fromdesiccated firefly tails. The firefly tails are first ground to a finepowder with a mortar and pestle with a small amount of washed silica.The powder is then extracted with 0.05 M AsO 0.0l M MgSO at pH 7.4.

The reaction mixture incorporating individual constituents is acontrolled mixture of luciferin, purified luciferase and a Mg**salt.This mixture may be prepared by dissolving luciferin, purifiedluciferase and MgSO. in a sterile aqueous solution. Aresenate and/orother buffers may be added to provide a pH 7.4.

In order to observe and record small amounts of light produced by apositive response between the material to be assayed and the reactionmixture and to make quantitative measurements of the light emitted,instruments which will sense and record the intensity of the emittedlight may be used. One

procedure consists of injecting the aqueous ATP extract prepared inaccordance with the practice of this invention into a cuvette containingthe reaction mixture. (Alternatively, the reaction mixture may beinjected into a cuvette containing the extract.) The extract is held atpH 7.4 with potassium arsenatc buffer. The light emitted as the resultof the reaction between any ATP in the aqueous extract and the reactionmixture strikes the photosensitive surface of a photomultiplier tubegiving rise to an electric signal which can be measured and recorded byeither an oscilloscope photograph or a chart recorder.

Because the response (i.e., light emission) is almost instantaneous, thereaction mixture should be positioned in front of the light detectorprior to the introduction of the extract. Also, the reaction mixture andthe extract should be mixed as rapidly as possible. The bioluminescentresponse with ATP is determined by measuring the maximum intensity ofthe emitted light, which after reaching this maximum value, decayslogarithmically. With all other factors constant, the maximum intensityis directly proportional to the concentration of ATP.

instrumentation useful for the quantitative measurement ofbioluminescence may consist of a photomultiplier tube for the conversionof light energy into an electrical signal, a device for determining themagnitude of the signal, and a light-tight chamber for presentation ofthe bioluminescent reaction to photomultiplier tube.

in one system, part of the assembly consists of a composite sensing andreaction chamber which contains a photomultiplier tube, with appropriatecircuitry, and a rotary cylinder mounted in a block of aluminum in amanner which permits removal of the reaction chamber without exposingthe phototube to light. A section of the cylinder wall is cut out toaccommodate a cuvette in a suitable reflector. Immediately above thecuvette holder is a small injection port sealed with a replaceablelight-tight rubber plug. The entire unit is painted black to reducelight reflection. The photomultiplier converts the light energy into anelectrical signal. An oscilloscope, which records the magnitude of thesignal from the photomultiplier, is provided with an adjustable verticaldeflection scale which will allow an adjustment in system sensitivity.There is a multiple switching arrangement at the oscilloscope inputwhich makes it convenient to adjust the system zeros and balances. thedifferential input to the oscilloscope provides a means to balance thedark current output of the phototube. The response to the fireflyluminescent system displayed on the oscilloscope screen is recorded witha camera which mounts directly onto the front of the oscilloscope. Toobserve and record the reaction, the cuvette containing the necessaryreagents is positioned in the cuvette holder without exposing thephototube. Rotation of the holder positions the cuvette in front of thephototube. The extract, presumed to contain ATP, is then added throughthe injection port and the magnitude ofthe response, if any, is recordedby the camera.

In order to make quantitative determinations of the amount of ATPpresent, the instrument used to measure the light response may becalibrated using known concentrations of ATP. A calibration may beplotted by injecting 1/l,000 ml. portions of known concentrations of ATPthrough the lightproof seal into the cuvette by means of a hypodermicsyringe. The light response is plotted against the ATP concentration. Alinear function is obtained.

For experimental purposes thirteen species of bacteria, likely to beencountered in contaminated environments, were cultured and samplestaken for analysis by the present method. The following procedure wasused for each bacterial culture:

I. A 2 ml. aliquot of each sample was filtered. through a microporousbacterial membrane;

2. The bacterial cells retained on the membrane were washed with 2 ml.of sterile cold 0.02 M AS0413 buffer;

3. The washed bacterial cells were extracted with a cold mixture of 1ml. of0.02 M A50 buffer /5 ml. n-butanol;

4. The volume of the aqueous phase was recorded;

5. A 0.01 ml. sample of the aqueous phase was injected into a reactionchamber containing a reaction mixture;

6. The total intracellular ATP was calculated from the response oflightemitted by the reaction;

7. The total number of cells in another aliquot of the same culture wasdetermined by plate counting, correcting for cell clumping, and

8. The total ATP per sample, in micrograms, was divided by the totalnumber of cells per the same size sample to obtain the ATP/cell for thebacterial culture.

The reaction mixture was prepared by combining 20 mg. luciferasedissolved in 2 ml. cold sterile water and 1.2 mg. luciferin dissolved in2 ml. cold 0.02 M AsO buffer (pH 7.4), and adding 2 ml. of0.0l M MgSOThe resultant data showed that a very high correlation existed betweenthe level of ATP in a sample and the number of bacterial cells of thespecies present. Totally unexpected was the important discovery thatintracellular ATP content remained relatively constant regardless ofspecies or growth phase.

A computer print out of this data is found in the drawing. Each numberon the printout indicates the number of data points occupying thatposition. By subsequent claculation it was found that the statisticalcorrelation between log ATP and log bacterial cells was 0.924. Over thethirteen species a mean value of about 5X10 pg. ATP/cell was observed.Each of the species is listed below with its corresponding calculatedvalue of intracellular ATP. (Note that each ATP value is within a :2fold variation of the mean.)

Bacterial Species Lactobacillu: careii g ATPXN) per cell To test theapplicability of this method to bacteria, other than those existing inpure cultures, comparisons were made between the present method and theclassical agar plate count method. By way of illustration arepresentative protocol for a bacteriuria assay will be given:

1. A 10 ml. aliquot ofa cold urine sample was centrifuged at 225xg(1,000 rpm. at 5%inches radius) for 5 minutes;

2. 2 ml. of the supernatant was filtered through a microporous bacterialmembrane;

3. The bacterial cells retained on the membrane were washed with 2 ml.of sterile cold saline;

4. The washed bacterial cells were extracted with a cold mixture of 1ml. 0.02 M AsO, buffer/S ml. n-butanol;

5. The volume of the aqueous phase was recorded;

6. A 0.01 ml. sample of the aqueous phase was injected into a reactionchamber containing the reaction mixture (prepared as in the previousexample);

7. The total ATP extracted was calculated from the response of lightemitted by the reaction; and

8. The number of bacterial cells was calculated using the formula X=Y/5l0 where X is the number of bacterial cells and Y is the quantity of ATPextracted, measured in micrograms.

The following table is a statistical comparison of bacterial countsobtained for various environments by this type of protocol and thatobtained by the classical agar plate count.

Milk 20 1. 248 M 0. 054 1501. 3'0 1.15-1.35 Fuel 12 0. 949 0.1a .s2-1.0s.69-1. 21 Pure cultures 1. 009 068 94-1. 08 871. 14

Correlatiorraiio ratio ;f instrumental ATl bacteria count to agar platecount for bacterial samples. 7

n =number of tests performed to determine the correlation ratio. Ludoxis a registered trademark for colloidal silica.

The bacterial counts obtained by utilizing the present invention werefound to be more valid than those obtained by the traditional platingmethod. An advantage of the ATP counts, in addition to ease and speed ofmeasurement, was the freedom from errors due to clumping of thebacterial cells or motility of some species; both of which can stronglyinfluence plate counts. The instrumental counts obtained by the processof this invention were shown to be true counts of individual bacteriaand could detect as few as 1,000 organisms in less than 5 minutes.

It will be understood that various changes in the details, materials,steps, order of steps, etc., which have been herein described andillustrated in order to explain the nature of the invention, may be madeby those skilled in the art within the principle and scope of theinvention as expressed in the appended claims.

I claim:

1. A method for determining the population density of bacterial cells ina measured sample of an aqueous suspension, said sample beingsubstantially devoid of interfering nonbacterial cells, said bacterialcells being in an unknown growth phase or of unknown species comprisinga. removing substantially all extracellular water-soluble material fromsaid measured sample of said suspension whereby substantially allbacterial cells are isolated from said suspension,

b. extracting adenosine triphosphate from said isolated bacterial cells,

c. measuring the quantity of adenosine triphosphate extracted, and d.determining the population density of bacterial cells from a singlepredetermined substantially constant relationship of quantity ofadenosine triphosphate to number of bacterial cells.

2. The method of claim 1 wherein said relationship of quantity ofadenosine triphosphate to number of bacterial cells is a constant ratio.

3. The method of claim 2 wherein said constant ratio has the value of5X10" microgram adenosine triphosphate per bacterial cell.

4. The method of claim 2 wherein substantially all of said bacterialcells are isolated from said suspension by passing said suspensionthrough a bacterial membrane filter.

5. The method of claim 2 wherein the quantity of said adenosinetriphosphate extracted is measured by reacting said adenosinetriphosphate with a mixture comprising luciferin and luciferase in thepresence of a metal cation and oxygen and measuring the light emitted.

6. The method of claim 5 wherein said cation is magnesium ion.

7. A method for the determination of the quantity of bacterial cellspresent in a sample of an aqueous suspension said sample beingsubstantially devoid of interfering nonbacterial cells, said cells beingin an unknown growth phase or of unknown species comprising a.separating substantially all bacterial cells from said sample,

b. extracting adenosine triphosphate from said separated bacterialcells,

c. measuring the quantity of adenosine triphosphate extracted and d.determining the number of bacterial cells in said sample,

from the formula X=Y 5 l0 where X is the number of said bacterial cellsand Y is the quantity of said adenosine triphosphate extracted, measuredin micrograms.

8. The method of claim 7 wherein the quantity of said adenosinetriphosphate extracted is measured by reacting said adenosinetriphosphate with a mixture comprising luciferin and luciferase in thepresence of a metal cation and oxygen and measuring the lightemitted. A

9. The met 0d of claim 8 wherein said cation IS magnesium ion.

Paw-1050 Patent No 3 616 253 Inventor(s) Dated October 26. 1971 AnthonvJ. D'Eustachio It is certified that error appears in theabove-identified patent and that said Letters Patent are herebycorrected as shown below:

[ Abstract,

Column Column Column Column 4,

Column t,

Column I,

Column L,

Claim 3, line 2, change "51110 to line 13,

line 39,

line 6H,

line

line

line

line

line

line

line

line

change change change before change change change change change changechange "5X1o to 5xlO' H II an to --on--.

"photomultiplier" insert --the--.

"AsOu to ASOL| 3 "AS0 to As0 "AS0 to A30 "5xlO to 5x10 "ATPxlo to--ATPxlO" "AS01413" to AS0L| 3 "y/5xl0 to --Y/5x1o- Claim 7, line 13,change "Y 5xlO to Y/5xl0' Signed and sealed this 13th day of June 1972.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. Attesting Officer ROBERT GOTTSCHALK Commissionerof Patents

2. The method of claim 1 wherein said relationship of quantity ofadenosine triphosphate to number of bacterial cells is a constant ratio.3. The method of claim 2 wherein said constant ratio has the value of 5X 10 10 microgram adenosine triphosphate per bacterial cell.
 4. Themethod of claim 2 wherein substantially all of said bacterial cells areisolated from said suspension by passing said suspension through abacterial membrane filter.
 5. The method of claim 2 wherein the quantityof said adenosine triphosphate extracted is measured by reacting saidadenosine triphosphate with a mixture comprising luciferin andluciferase in the presence of a metal cation and oxygen and measuringthe light emitted.
 6. The method of claim 5 wherein said cation ismagnesium ion.
 7. A method for the determination of the quantity ofbacterial cells present in a sample of an aqueous suspension said samplebeing substantially devoid of interfering nonbacterial cells, said cellsbeing in an unknown growth phase or of unknown species comprising a.separating substantially all bacterial cells from said sample, b.extracting adenosine triphosphate from said separated bacterial cells,c. measuring the quantity of adenosine triphosphate extracted and d.determining the number of bacterial cells in said sample, from theformula X Y/5 X 10 10, where X is the number of said bacterial cells andY is the quantity of said adenosine triphosphate extracted, measured inmicrograms.
 8. The method of claim 7 wherein the quantity of saidadenosine triphosphate extracted is measured by reacting said adenosinetriphosphate with a mixture comprising luciferin and luciferase in thepresence of a metal cation and oxygen and measuring the light emitted.9. The method of claim 8 wherein said cation is magnesium ion.